Ahmed I. El-Sakka

PEYRONIE'S DISEASE IS ASSOCIATED WITH AN INCREASE IN TRANSFORMING GROWTH FACTOR-beta PROTEIN EXPRESSION

PURPOSE Transforming growth factor-beta (TGF-beta) has been implicated in many chronic fibrotic conditions such as pulmonary and hepatic fibrosis. We postulated that TGF-beta may play a role in the pathogenesis of Peyronies disease. MATERIALS AND METHODS Tissues from the tunica albuginea of 30 Peyronies disease patients (study group) and from 6 patients without Peyronies disease, who had undergone penile prosthesis surgery for organic impotence (control group), were subjected to histological examination using Hart and trichrome stains and Western blotting for the detection of TGF-beta protein expression. RESULTS The results of these experiments demonstrate that all tissue from Peyronies disease patients showed a variety of histological changes of the tunica, ranging from chronic inflammatory cellular infiltration to complete calcification and ossification of the tissues. The most prominent changes observed in the majority of patients were focal or diffused elastosis, fenestration and disorganization of the collagen bundles. TGF-beta1 protein expression was detected in 26 patients (86%), while only 7 (23%) and 5 (17%) patients showed TGF-beta2 and TGF-beta3 protein expression, respectively. One patient in the control group showed fibrosis of the tunica albuginea and protein expression of TGF-beta1 and TGF-beta2. This patient had undergone surgery for the revision of his prosthesis twice. Five patients from the control group showed normal histological patterns of the tunica albuginea and no protein expression for TGF-beta1, TGF-beta2 and TGF-beta3. CONCLUSIONS TGF-beta1 protein expression is significantly associated with Peyronies disease, which may provide a new insight and the potential for the prevention and treatment of this disease.

VENOUS PATCH GRAFT FOR PEYRONIE'S DISEASE. PART I: TECHNIQUE

PURPOSE We describe our technique of plaque incision and venous patch grafting to correct complex penile deformity associated with Peyronies disease. MATERIALS AND METHODS Graft material is obtained from the lower and upper saphenous and deep dorsal veins. The configuration, size and number of tunical incisions depend on the site and size of the lesion. In most cases an H-shaped tunical incision is adequate to release the contracture. With the aid of a vascular stapler several vein segments can be assembled easily into 1 piece to cover the defect. RESULTS The incidence of penile shortening and erectile dysfunction, following other corrective procedures, is lessened with the tunical incision and venous grafting technique. CONCLUSION The venous graft provides an anatomical and functional tunical substitute. Results in correction of Peyronies disease are highly encouraging.

AN ANIMAL MODEL OF PEYRONIE'S-LIKE CONDITION ASSOCIATED WITH AN INCREASE OF TRANSFORMING GROWTH FACTOR BETA mRNA AND PROTEIN EXPRESSION

PURPOSE Transforming growth factor beta (TGF-beta) is involved in numerous vital processes including tissue fibrosis. Our objective was to study the role of TGF-beta in the induction of a Peyronies-like condition and to produce an animal model for the further study of Peyronies disease. MATERIALS AND METHODS Twenty-four adult male Sprague-Dawley rats were divided into two groups. Different concentrations of cytomodulin, a synthetic heptopeptide with TGF-beta-like activity, were injected into the tunica of each rat from the first group (n = 18). Rats in the second group (n = 6) received saline injections as a control. The tunical tissues were taken after 3 days, 2 weeks, and 6 weeks and were examined using Hart and Trichrome stains. In the same tissue samples, TGF-beta mRNA and protein expression were studied. RESULTS Histological alterations were observed in 15 out of 18 cytomodulin-injected rats, especially in tissue examined after 6 weeks. The most prominent changes were chronic cellular infiltration, focal and diffuse elastosis, thickening, disorganization and clumping of the collagen bundles. Results from immunoblot revealed remarkable TGF-beta1 protein expression in all the cytomodulin-injected rats only after 2 and 6 weeks. No remarkable TGF-beta2 or TGF-beta3 protein expression was observed. TGF-beta1 mRNA expression in the cytomodulin-injected rats was noticed in rats injected with higher concentrations after 3 days, while it was expressed in all rats after 2 weeks. There was no expression in the control group after either 3 days or 2 weeks. CONCLUSIONS Cytomodulin can induce Peyronies-like condition in the rat penis, which may explain the role of TGF-beta in the pathogenesis of Peyronies disease.

VENOUS PATCH GRAFT FOR PEYRONIE'S DISEASE. PART II: OUTCOME ANALYSIS

PURPOSE We evaluate the results of tunical incision and venous patch grafting for correcting penile deformity in Peyronies disease. MATERIALS AND METHODS In 113 [corrected] patients with symptoms of Peyronies disease for more than a year indications for surgery included penile shortening, persistent pain, severe curvature, penile narrowing or indentation and/or failure of previous surgery. Preoperative evaluation included determination of patient and partner expectation, potency status, circumcision status, measurement of penile length (short and long side) and saphenous vein, and color duplex ultrasonography to evaluate possible accessory vascular communication. Patients underwent plaque incision and venous patch grafting. The configuration, size and number of tunical incisions depended on the size and shape of the lesion. Lower and upper saphenous, and deep dorsal veins served as the graft materials. Postoperative followup was as long as 18 months. RESULTS In 96% of patients the penis became straight, while residual curvature was 30 degrees in 3% and 15 degrees in 1%. In 94% of patients narrowing and indentation were absent and in 83% penile length was the same or longer postoperatively. Of the patients who were potent preoperatively 88% experienced the same or better erectile quality after surgery. In 10% of cases a change in sensation occurred lasting longer than 6 months. Overall satisfaction was expressed by 92% of men who believed that surgery improved the psychological state as well as the relationship with the partner. CONCLUSIONS The results are satisfactory and this procedure offers a reasonable solution for correction of Peyronies disease.

The effect of vascular endothelial growth factor on a rat model of traumatic arteriogenic erectile dysfunction

PURPOSE We tested the hypothesis that intracavernous injection of vascular endothelial growth factor (VEGF) can restore erectile function in a rat model of traumatic arteriogenic erectile dysfunction. MATERIALS AND METHODS Exploration of bilateral internal iliac arteries was performed in 50, 3-month-old male rats. A total of 44 rats underwent bilateral ligation of the internal iliac arteries and 6 that underwent exploration only served as the sham operated group. Minutes later intracavernous injection of phosphate buffered saline (PBS) plus bovine serum albumin in 16 rats, 2 microg. VEGF plus PBS plus BSA in 12 and 4 microg. VEGF plus PBS plus BSA in 16 was performed. At weeks 1, 2 and 6 about a third of the rats in each group underwent electrostimulation of the cavernous nerves to assess erectile function and were then sacrificed. Penile tissues were collected for histochemical and electron microscopy examinations. RESULTS No impairment of erectile function was noted in sham operated rats. Immediately after arterial ligation all rats showed little or no erectile response to neurostimulation. In PBS treated rats modest recovery of erectile function was noted at week 6. Significant recovery of erectile function was noted in VEGF treated rats at weeks 1 and 2 in the 4 microg. group only and at week 6 in the 2 and 4 microg. groups. Neuronal nitric oxide synthase staining showed a reduction in neuronal nitric oxide synthase positive nerve fibers in the dorsal or intracavernous nerves at week 1. Moderate recovery of neuronal nitric oxide synthase positive nerve fibers was noted in the 2 and 4microg. VEGF treated groups but not in the PBS treated group. Electron microscopy revealed no pathological change in sham operated rats. In dorsal nerves the atrophy of myelinated and nonmyelinated nerve fibers was noted in ligated plus PBS treated rats. Partial recovery was observed in VEGF treated rats. Scattered atrophic smooth muscle cells were seen in PBS and occasionally in VEGF treated rats but not in the sham operated group. The most dramatic findings in VEGF treated rats were hypertrophy and hyperplasia of the endothelial cells, especially those lining the small capillaries. CONCLUSIONS Ligation of bilateral internal iliac arteries produced a reliable animal model of traumatic arteriogenic erectile dysfunction. Intracavernous injection of VEGF minutes after arterial ligation facilitated the recovery of erectile function.

LENGTHENING SHORTENED PENIS CAUSED BY PEYRONIE'S DISEASE USING CIRCULAR VENOUS GRAFTING AND DAILY STRETCHING WITH A VACUUM ERECTION DEVICE

PURPOSE We evaluated the results of chronic intermittent stretching with a vacuum erection device after circumferential tunical incision and circular venous grafting in 4 patients with penile shortening from severe Peyronies disease. MATERIALS AND METHODS We performed complete circumferential tunical incision and covered the defect with a circular venous graft in 4 patients with shortened penis as a result of Peyronies disease. Preoperative evaluation included determination of patient and partner expectations, potency status, measurement of penile length after intracavernous injection and color duplex ultrasonography to determine possible vascular communication. Lower saphenous, upper saphenous and deep dorsal veins served as graft materials. We advised patients to use a vacuum device on a daily basis for 6 months starting 1 month after surgery. Postoperative evaluations were done at 6 and 18 months postoperatively. RESULTS At 6-month followup 1 patient who did not use the vacuum device gained 1 inch in penile length and was not available for further followup. The other 3 patients each gained 2 inches but had decreased erectile rigidity due to narrowing in the grafted area (hourglass deformity). One patient who wanted a more natural erection elected penile prosthesis implantation about 1 year after grafting. The remaining 2 patients gained 3 inches at 18-month followup and regained partial penile rigidity similar to preoperative erections when the hourglass deformity improved. All patients were satisfied and indicated that surgery improved psychological well-being as well as relationships with partners. CONCLUSIONS The results in this small group are satisfactory. Our technique offers a reasonable solution for correction of penile shortening in patients with Peyronies disease.

EFFECT OF CAVERNOUS NERVE FREEZING ON PROTEIN AND GENE EXPRESSION OF NITRIC OXIDE SYNTHASE IN THE RAT PENIS AND PELVIC GANGLIA

PURPOSE Cryoablation of the prostate has been reported to induce impotence as a result of cavernous nerve injury. This study is designed to investigate the early and late effects of cavernosal nerve cryoablation on erectile function and nitric oxide synthase (NOS) containing nerves in the rat penis and pelvic ganglia. MATERIALS AND METHODS Forty male rats were divided into two groups (n = 20 each). The first group underwent unilateral cavernous nerve freezing (experimental group). Before their euthanization at 1 and 3 months (10 rats each), erectile function was assessed by electrostimulation of the cavernous nerves. The second group served as a control and was euthanized at the same time points. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining was used to identify NOS in the penile nerve fibers of the mid-shaft segment and neurons of the pelvic ganglia. Western blot, RT-PCR and immunostaining techniques were used to identify protein and gene expression of eNOS, iNOS, and nNOS. RESULTS One month after unilateral cavernosal nerve freezing, there was a significant decrease in NOS-containing nerve fibers in the dorsal and intracavernosal nerves and ipsilateral pelvic ganglia as compared with the intact side. At three months, the number of NOS containing nerve fibers in the above-mentioned nerves and ganglia showed a significant increase on the frozen side but not the intact side. Electrostimulation of the frozen nerve after three months revealed a significantly higher maximal intracavernosal pressure and a shorter latency period than the one-month group. At three months, immunoblot showed up-regulation of eNOS and nNOS protein expression in the penile tissue. RT-PCR showed up-regulation of nNOS gene expression in the pelvic ganglia of the frozen side. Immunostaining confirmed the results of western blot and showed significant increase of the nNOS positive staining in the frozen side of the penile tissue after three months. There was no difference in iNOS after three months between both sides. Our repeated eNOS immunostaining was not successful. CONCLUSIONS The results show that intracavernous pressure response to neurostimulation markedly decreased at one month and then partially recovered three months after cavernosal nerve freezing. A similar pattern of changes of the NOS-containing nerve fibers in dorsal nerves, intracavernosal nerves, and pelvic ganglia were observed. This alteration in erectile function and NOS-containing nerves was associated with differential gene and protein expression of the three subtypes of NOS.

THE EFFECTS OF COLCHICINE ON A PEYRONIE'S-LIKE CONDITION IN AN ANIMAL MODEL

PURPOSE We have developed an animal model of Peyronies disease by injecting transforming growth factor beta (TGF-beta) into the rat penis. Our objective is to study the effects of colchicine on the Peyronies condition in an animal model. MATERIALS AND METHODS Thirty-six adult male Sprague-Dawley rats received TGF-beta injections into the tunica albuginea and were divided into two groups (n = 18 each). Rats in the first group were divided into three subgroups (n = 6 each). Each rat in the three subgroups received the following: Subgroup 1 received colchicine, subgroup 2 received ibuprofen, and subgroup 3 received regular water. The rats were euthanized after 6 weeks. Rats in the second group were also divided into three subgroups. These rats received the same treatments as the rats in the first group, but treatments began 6 weeks after TGF-beta injection. These rats were euthanized after 12 weeks. Tunical tissue samples were collected and examined using Hart and trichrome stains, electron microscopy (EM), and western blot analysis for TGF-beta detection. RESULTS In the first group, the colchicine-treated rats exhibited less collagen deposition and less elastic fiber fragmentation than the untreated or ibuprofen-treated rats. EM confirmed the results and showed normal distribution and shape of both collagen and elastic fibers in the colchicine-treated group. In the second group, the colchicine-treated rats exhibited less crowding of the collagen fibers. However, the elastic fibers remained fragmented and scarce. Western blot analysis showed significant down-regulation of TGF-beta expression (5/6) in the colchicine-treated group after 6 weeks. Down-regulation was observed in only 1/6 in both ibuprofen and non-treated groups. After 12 weeks 2/6, 1/6, and 1/6 rats displayed down regulation in the colchicine treated, ibuprofen treated, and non-treated groups, respectively. CONCLUSION Early colchicine treatment may suppress a Peyronies like condition in the rat animal model.

Effect of a Chinese herbal medicine mixture on a rat model of hypercholesterolemic erectile dysfunction

PURPOSE We examine the effect of a Chinese herbal medicine mixture on erectile function in a rat model of hypercholesterolemic erectile dysfunction. MATERIALS AND METHODS In this study 32, 3-month-old Sprague-Dawley rats were used. The 8 control animals were fed a normal diet and the remaining 24 were fed 1% cholesterol diet for 4 months. After 2 months herbal medicine was added to the drinking water of the treatment group of 16 rats but not the cholesterol only group of 8. Of the 16 rats 8 received 25 mg./kg. per day (group 1) and 8 received 50 mg./kg. per day (group 2) of Chinese herbal medicine mixture. Serum cholesterol levels were measured at 2 and 4 months. At 4 months erectile function was evaluated with cavernous nerve electrostimulation in all animals. Penile tissues were collected for electron microscopy, and to perform Western blot for endothelial nitric oxide synthase, neuronal nitric oxide synthase, basic fibroblast growth factor (bFGF) and caveolin-1. RESULTS Serum cholesterol levels were significantly higher in animals fed the 1% cholesterol diet compared to controls at 2 and 4 months. Nevertheless, there was no significant difference among group 1 (145 +/- 30 mg./dl.), group 2 (157 +/- 20) and the cholesterol only group (143 +/- 15). Systemic arterial pressure was not significantly different between the animals that were fed the 1% cholesterol diet and the controls. During electrostimulation of the cavernous nerve peak sustained intracavernous pressure was significantly lower in the cholesterol only group (50 +/- 23 cm. H2O) compared to the control group. Conversely erectile function was not impaired in the herbal medicine treated rats. Electron microscopy showed many caveolae with fingerlike processes in the cavernous smooth muscle and endothelial cell membranes in control and treated rats but not in the cholesterol only group of rats. Western blot did not show a difference among groups in protein expression for endothelial nitric oxide synthase and neuronal nitric oxide synthase in penile tissue but caveolin-1 and bFGF protein expression was significantly higher in groups 1 and 2 than in the cholesterol only and control groups. CONCLUSIONS Rats developed erectile dysfunction after being fed a 1% cholesterol diet for 4 months. Although serum cholesterol levels were similar in the cholesterol only rats and those treated with Chinese herbal medicine mixture, erectile response was significantly better in the treated group. The mechanism of the herbal medicine is unknown. High levels of bFGF and caveolin-1 expression in the treated group may protect the cavernous smooth muscle and endothelial cells from the harmful effect of high serum cholesterol.

EXPRESSION OF TGF-beta-1 mRNA AND ULTRASTRUCTURAL ALTERATIONS IN PHARMACOLOGICALLY INDUCED PROLONGED PENILE ERECTION IN A CANINE MODEL

PURPOSE Transforming growth factor beta (TGF-beta) is known to induce fibrosis. Our objective was to study the role of TGF-beta as a possible mediator of fibrosis that may follow prolonged penile erection. MATERIALS AND METHODS Prolonged penile erection was induced in seven adult male mongrel dogs by intracavernosal injection of papaverine into one of the corpora cavernosa while the other was used as a control. Intracavernosal pressure measurements were carried out prior to administration of papaverine and at the end of the procedure. Penile tissue was collected from anesthetized animals prior to euthanasia for histological and electron microscopic (EM) studies. RT-PCR was carried out for detection of mRNA on same tissue samples. RESULTS The light microscopy showed stasis of blood in the cavernosal sinusoids. EM studies revealed sporadic endothelial defects, loss of plasma membrane integrity and cytoplasmic condensation. There was expression of TGF-beta1 mRNA in 66.7% of the experimental group compared with 16.7% of the control group. CONCLUSIONS Pharmacologically induced low flow prolonged penile erection in canine models is associated with histomorphological changes in relatively short periods of time, suggesting that early therapeutic intervention is desirable. The gene expression for TGF-beta1 may be a mediator of fibrosis; therefore the use of anti-TGF-beta agents presents a possible tool for therapeutic intervention.